In animals and in the first human studies, our group showed that a single dose of AAV-CFTR could be applied to the nasal epithelium, right lower lobe of the human lung, and the maxillary sinus, without any adverse effects. These studies and several other animal studies published in the literature showed that gen expressed from AAV vectors result in persistent expression in a variety of tissues. Thus, AAV vectors have great potential as therapeutic agents. Despite these promising studies, several hurdles must be overcome before AAV vectors will be useful therapeutic agents. This grant will use a combination of animal and human studies to address the hypothesis that repeated delivery of AAV-CFTR vectors via aerosol delivery to the airways will be safe and result in increased levels of recombinant CFTR expression. There are three overall questions that will be addressed. 1. Will repeat dosing with AAV-vectors lead to increased expression of the recombinant vector? This aim is designed to determine the effects of repeated dosing of AAV-vectors on transgene expression, in a rabbit bronchoscopy model. It is anticipated that answers to this question will be critical in determining whether modification of vector capsid proteins and/or interventions in the immune response will be necessary before repeated delivery will lead to successful gene expression. 2. Is aerosol delivery of AV-CFTR a practical alternative to bronchoscopic delivery? It is anticipated that answers to this question in a non-human primate model will provide the pre-clinical data necessary to launch a study of aerosolized AAV-CFTR vector in patients with CF. 3. Does aerosol delivery of higher titer AAV-CFTR vectors administered in CF patients with Mild Lung Disease lead to widespread gene transfer and CFTR expression? Particular attention will also be given to whether repeat dosing for AAV-CFTR in humans leads to increased expression of the recombinant vector. It is expected that answers to this question will lead to a practical delivery system for recombinant AAV-CFTR.